1. Field of the Invention
This invention relates generally to the modulation of neural growth in the central nervous system, and more particularly to methods and associated agents, constructs and compositions for improving CNS neural growth. Specifically, the invention relates to the use of cellular adhesion molecules, and preferably neural cell adhesion molecules such as L1, to foster and improve such neural growth.
2. Description of the Related Art
The ability of neurons to extend neurites is of prime importance in establishing neuronal connections during development. It is also required during regeneration to re-establish connections destroyed as a result of a lesion.
Neurites elongate profusely during development both in the central and peripheral nervous systems of all animal species (Cajal (1928) Degeneration and regeneration in nervous system, Oxford University Press, London). This phenomenon pertains to axons and dendrites. However, in adults, axonal and dendritic regrowth in the central nervous system is increasingly lost with evolutionary progression.
In the peripheral nervous system, after infliction of a lesion, axons of all vertebrate species are able to regrow (Cajal (1928); Martini (1994) J. Neurocytol. 23:1-28). However, in mammals, neurite regrowth following damage is limited to neuritic sprouting. Regrowth of neuronal processes is, however, possible in lower vertebrate species (Stuermer et al. (1992) J. Neurobiol. 23:537-550). In contrast, in the central nervous system, most, if not all neurons of both higher and lower vertebrate adults possess the potential for neurite regrowth (Aguayo (1985) xe2x80x9cAxonal regeneration from injured neurons in the adult mammalian central nervous system,xe2x80x9d In: Synaptic Plasticity (Cotman, C. W., ed.) New York, The Guilford Press, pp. 457-484.)
Glial cells are the decisive determinants for controlling axon regrowth. Mammalian glial cells are generally permissive for neurite outgrowth in the central nervous system during development (Silver et al. (1982) J. Comp. Neurol. 210:10-29; Miller et al. (1985) Develop. Biol. 111:35-41; Pollerberg et al. (1985) J. Cell. Biol. 101:1921-1929) and in the adult peripheral nervous system (Fawcett et al. (1990) Annu. Rev. Neurosci 13:43-60). Thus, upon infliction of a lesion, glial cells of the adult mammalian peripheral nervous system can revert to some extent to their earlier neurite outgrowth-promoting potential, allowing them to foster regeneration (Kalderon (1988) J. Neurosci Res. 21:501-512; Kliot et al. xe2x80x9cInduced regeneration of dorsal root fibres into the adult mammalian spinal cord,xe2x80x9d In: Current Issues in Neural Regeneration, New York, pp. 311-328; Carlstedt et al. (1989) Brain Res. Bull. 22:93-102). Glial cells of the central nervous system of some lower vertebrates remain permissive for neurite regrowth in adulthood (Stuermer et al. (1992) J. Neurobiol. 23:537-550). In contrast, glial cells of the central nervous system of adult mammals are not conducive to neurite regrowth following lesions.
Several recognition molecules which act as molecular cues underlying promotion and/or inhibition of neurite growth have been identified (Martini (1996). Among the neurite outgrowth promoting recognition molecules, the neural cell adhesion molecule L1 plays a prominent role in mediating neurite outgrowth (Schachner (1990) Seminars in the Neurosciences 2:497-507). L1-dependent neurite outgrowth is mediated by homophilic interaction. L1 enhances neurite outgrowth on L1 expressing neurites and Schwann cells, and L1 transfected fibroblasts (Bixby et al. (1982) Proc. Nat""l Acad. Sci. U.S.A. 84:2555-2559; Chang et al. (1987) J. Cell. Biol. 104:355-362; Lagenaur et al. (1987) Proc. Natl. Acad. Sci. USA 84:7753-7757; Seilheimer et al. (1988) J. Cell. Biol. 107:341-351; Kadmon et al. (1990a) J. Cell. Biol. 110:193-208; Williams et al. (1992) J. Cell. Biol. 119:883-892). Expression of L1 is enhanced dramatically after cutting or crushing peripheral nerves of adult mice (Nieke et al. (1985) Differentiation 30:141-151; Martini et al. (1994a) Glia 10:70-74). Within two days L1 accumulates at sites of contact between neurons and Schwann cells being concentrated mainly at the cell surface of Schwann cells but not neurons (Martini et al. (1994a)). Furthermore, the homophilic binding ability of L1 is enhanced by molecular association with the neural cell adhesion molecule N-CAM, allowing binding to occur through homophilic assistance (Kadmon et al. (1990a); Kadmon et al. (1990b) J. Cell Biol. 110:209-218 and 110:193-208; Horstkorte et al. (1993) J. Cell. Biol. 121:1409-1421). Besides its neurite outgrowth promoting properties, L1 also participates in cell adhesion (Rathjen et al. (1984) EMBO J. 3:1-10; Kadmon et al. (1990b) J. Cell. Biol. 110:209-218; Appel et al. (1993) J. Neurosci., 13:4764-4775), granule cell migration (Lindner et al. (1983) Nature 305:427-430) and myelination of axons (Wood et al. (1990) J. Neurosci 10:3635-3645).
L1 consists of six immunoglobulin-like domains and five fibronectin type III homologous repeats. L1 acts as a signal transducer, with the recognition process being a first step in a complex series of events leading to changes in steady state levels of intracellular messengers. The latter include inositol phosphates, Ca2+, pH and cyclic nucleotides (Schuch et al. (1990) Neuron 3:13-20; von Bohlen und Hallbach et al. (1992) Eur. J. Neurosci. 4:896-909; Doherty et al. (1992) Curr. Opin. Neurobiol. 2:595-601) as well as changes in the activities of protein kinases such as protein kinase C and pp60c-src (Schuch et al. (1990) Neuron 3:13-20; Atashi et al. (1992) Neuron 8:831-842). L1 is also associated with a casein type II kinase and another unidentified kinase which phosphorylates L1 (Sadoul et al. (1989) J. Neurochem 328:251-254). L1-mediated neurite outgrowth is sensitive to the blockage of L type Ca2+ channels and to pertussis toxin. These findings indicate the importance of both Ca2+ and G proteins in L1-mediated neurite outgrowth (Williams et al. (1992) J. Cell. Biol. 119:883-892). L1 is also present on proliferating, immature astrocytes in culture and neurite outgrowth is promoted on these cells far better than on differentiated, L1 immunonegative astrocytes (Saad et al. (1991) J. Cell. Biol. 115:473-484). In vivo, however, astrocytes have been found to express L1 at any of the developmental stages examined from embryonic day 13 until adulthood (Bartsch et al. (1989) J. Comp. Neurol 284:451-462; and unpublished data).
Given the capability of L1 to promote neurite outgrowth, it is pertinent to investigate whether astrocytic expression of L1 and other members of the immunoglobulin superfamily to which L1 belongs, may overcome potentially inhibitory molecular cues reported to be present on glial cells and myelin in the adult central nervous system (Schachner et al., Perspectives in Developm. Neurobiol. in Press; Schwab et al. (1993) Ann. Rev. Neurosci. 16:565-595). This is of particular relevance to the development of effective strategies for the treatment of debilitation caused by the malformation of or injury to neural tissues of the CNS, and it is toward such objectives that the present invention is directed.
In accordance with the present invention, an agent and corresponding methods are disclosed for the modulation of neural growth and particularly, such growth as can be promoted in the compartment of the central nervous system (CNS), and specifically, in myelinated nerve tissue. The agents of the present invention are notable in their ability to promote such neural growth in an environment that has been traditionally viewed as inhibitory to the growth promoting stimulus of known neurite outgrowth factors. Specifically, this inhibitory environment includes inhibitory molecular cues which are present on glial cells and myelin the central nervous system.
The agents of the present invention are broadly selected from a group of cell adhesion molecules, and more preferably neural cell adhesion molecules. Most preferably, the agents of the present invention are selected from the group of molecules belonging to the immunoglobulin superfamily, and particularly to those members that mediate Ca2+-independent neuronal cell adhesion, of which L1, N-CAM and myelin-associated glycoprotein are particular members. Other cell adhesion molecules which may also influence CNS neural growth include laminin, fibronectin, N-cadherin, BSP-2 (mouse N-CAM), D-2, 224-1A6-A1, L1-CAM, NILE, Nr-CAM, TAG-1 (axonin-1), Ng-CAM and F3/F11.
In a further aspect of the present invention, the agents of the invention belong to a new family referred to herein as the L1 family of neural recognition molecules. This family includes L1, NgCAM, neurofascin, Drosophila neuroglian, zebrafish L1.1 and L1.2, and others. This group of agents all demonstrate the Ig-like domains and FN-like repeats that are characteristic of L1, and in this connection, exhibit a remarkable colinearity, a high degree of N-glycosidically linked carbohydrates, which include the HNK-1 carbohydrate structure, and a pattern of protein fragments comprising a major 185 kD band and smaller bands of 165 and 125 kD.
The agents of the present invention also include fragments including soluble fragments, of cell adhesion molecules and cognate molecules, congeners and mimics thereof which modulate neurite growth in the CNS. In particular, the agents include molecules which contain structural motifs characteristic of extracellular matrix molecules, in particular the fibronectin type III homologous repeats and immunoglobulin-like domains. Preferably, these structural motifs include those structurally similar to fibronectin type III homologous repeats 1-2, and immunoglobulin-like domains I-II, III-IV and V-VI.
The invention extends to methods of promoting and enhancing neural regeneration including neural growth and/or remyelination and/or neuroprotection in vivo, and to the corresponding genetic constructs, such as plasmids, vectors, transgenes, and the like, and to pharmaceutical compositions, all of which may be used to accomplish the objectives of such methods. More specifically, the agents of the present invention may be prepared as vectors or plasmids, and introduced into neural cells located at a site in the CNS where such regeneration or other therapy is needed, for example, by gene therapy techniques, to cause the expression of an agent of the present invention and to thereby promote the requisite neural growth. Another strategy contemplates the formulation of one or more of the appropriate agents in a composition that may likewise be directly delivered to a CNS site, as by parenteral administration. As certain of the agents, such as L1, have demonstrated homophilic binding, the administration of such a composition may serve the purpose of inhibiting rather than promoting neural growth. This effect may be desirable in particular instances where unwanted or uncontrolled growth may occur or is occurring, and therefore the invention extends to this use as well.
Correspondingly, the capability of the agents to engage in homophilic binding renders antagonists to the agents, including antibodies thereto as well as soluble fragments thereof, capable of acting as agonists, and thereby participating in the promotion of neural growth and/or remyelination and/or neuroprotection and regeneration. Thus, the invention extends to the preparation of appropriate constructs and compositions containing the antibodies to the agents, and any soluble fragments, for the therapeutic purposes set forth herein. Also, and as demonstrated later on herein, antibodies to L1 and soluble fragments of L1, for example, may serve as part of a drug discovery assay or the like, to identify further agents that may possess activity and utility both diagnostic and therapeutic, in accordance with the present invention. Particularly, and as illustrated later on herein with reference to the isolation and characterization of CHL1, an L1 analog, antibodies such as polyclonal, monoclonal and chimeric (bispecific) antibodies, including humanized such antibodies, may be used to identify further members of the L1 CAM family, and the invention accordingly extends to such CAM members as are isolated by use of such antibodies.
The invention also covers diagnostic applications, where for example, it is desirable to assess the potential for or actual development of CNS neural growth by the detection and measurement of the presence, amount or activity of one or more of the agents of the invention. Likewise, and as described hereinafter, the invention also extends to assays, including drug discovery and testing assays, that capitalize on the activity of the agents of the present invention in the modulation of CNS neural growth. For example, prospective drugs may be tested for CNS neural growth modulation by means of an assay containing an agent of the invention, or a cell line or culture developed in conjunction herewith may serve as the assay system.
Briefly, the present invention also features transgenic mouse lines expressing a neural adhesion molecule in differentiated astrocytes and glial cells, and cells and tissues derived therefrom. In particular, the neural adhesion molecule is L1. The astroglial L1 expression enhances neurite outgrowth on central nervous system tissue derived from these transgenic mice.
Also as discussed, the invention features methods for enhancing neuronal outgrowth of CNS neurons, for enhancing memory and for increasing synaptic efficacy, as may be measured by stabilization of long term potentiation, and other similarly useful methods. A related aspect of the present invention extends to the neuroprotective effects that are exerted by the present agents including the neural adhesion molecules set forth herein. Among the consequences of such activity is the application of the present agents to the modulation of conditions where neuroprotective activity is relevant, including the reduction of apoptosis and necrosis, and the related effects on neurodegenerative disorders such as Alzheimers and Parkinsons Disease, and multiple sclerosis. Similarly, the agents are capable of increasing neuronal survival as is likewise beneficial in the treatment of the presence or consequences of Alzheimers"" disease and Parkinsonism, and are also associated with an increase in remyelination in the central nervous system, and thus would offer an additional strategy for the treatment of conditions where the myelin sheath is destroyed or damaged, such as multiple sclerosis. Accordingly, the present invention includes methods of promoting neuroprotection and/or neuronal survival in a mammal by the administration of an effective amount of the neural adhesion molecules of this invention and/or cells or other constructs that will promote and/or assist in realizing the objectives and benefits of the methods.
Also featured are methods of testing drugs and other manipulations which modulate the effects of the neural adhesion molecule, and assay systems suitable for such methods.
Accordingly, it is a principal object of the present invention to provide a transgenic mammal, the glial cells of which express an exogenous neural adhesion molecule.
A further object of the invention is to provide a cell culture containing the glial cells of the transgenic mammal.
Yet another object of the invention is to provide a cell culture system containing lesioned or unlesioned optic nerves or other parts of the nervous system of the transgenic mammal.
Still a further object of the invention is to provide a method for enhancing neuronal outgrowth of CNS neurons, which includes culturing the neurons on the glial cell culture system.
A further object of the invention is to provide a method for enhancing neuronal outgrowth of CNS neurons, which includes culturing the neurons on the optic nerve or other parts of the nervous system placed in the cell culture system.
A still further object of the invention is to provide a method for enhancing neuronal outgrowth of CNS neurons, which includes the secretion of neural adhesion molecule by implanted cells.
Another object of the invention is to provide a method for enhancing memory, which includes administering to the brain of a mammal, an amount of the cells of the glial cell culture system effective to enhance the memory of the mammal.
Yet another object of the invention is to provide a method for enhancing memory, including administering to the brain of a mammal, an amount of the cells of the optic nerve or other parts of the nervous system placed in the cell culture system effective to enhance the memory of the mammal.
A still further object of the invention is to provide a method for enhancing memory, including delivering to the glial cells of the brain of a mammal, including mammals in need of such memory enhancement, a vector which allows for the expression of a neural adhesion molecule in the glial cells.
A further object of the invention is to provide a method for enhancing memory, which includes the secretion of neural adhesion molecule by implanted cells.
In a yet further object of the present invention a method is provided for the treatment of neurodegenerative conditions such as Alzheimers disease and Parkinson""s disease, by the administration of one or more of the present agents.
In a still further object of the present invention a method is provided for the promotion of remyelination and the corresponding treatment of conditions such as multiple sclerosis, by the administration of one or more of the present agents.
Another object of the invention is to provide a method for increasing synaptic efficacy in the CNS of a mammal, including administering to the brain of the mammal, an amount of the cells of the glial cell culture system effective to increase synaptic efficacy in the brain of the mammal.
Yet a further object of the invention is to provide a method for increasing synaptic efficacy in the CNS of a mammal, including administering to the brain of the mammal, an amount of the cells of the optic nerve or other parts of the nervous system placed in the cell culture system effective to increase synaptic efficacy in the brain of the mammal.
A still further object is to provide a method for increasing synaptic efficacy in the CNS of a mammal, which includes delivering to the glial cells of the brain of a mammal in need of such enhancement, a vector which allows for the expression of a neural adhesion molecule in the glial cells.
A further object of the invention is to provide a method for increasing synaptic efficacy, which includes the secretion of neural adhesion molecule by implanted cells.
A still further object of the present invention extends to the treatment of conditions involving a reduction or dysfunction in synaptic efficiency, and includes the treatment of pre-senile and senile dementias.
Another object of the invention is to provide a method of testing the ability of a drug or other entity to modulate the activity of a neural adhesion molecule, which includes adding CNS neurons to the glial cell culture system; adding the drug under test to the cell culture system; measuring the neuronal outgrowth of the CNS neurons; and correlating a difference in the level of neuronal outgrowth of cells in the presence of the drug relative to a control culture to which no drug is added to the ability of the drug to modulate the activity of the neural adhesion molecule.
Another object of the invention is to provide a method of testing the ability of a drug or other entity to modulate the activity of a neural adhesion molecule which includes adding CNS neurons to the optic nerve or other parts of the nervous system cell culture system; adding the drug under test to the cell culture system; measuring the neuronal outgrowth of the CNS neurons; and correlating a difference in the level of neuronal outgrowth of cells in the presence of the drug relative to a control culture to which no drug is added to the ability of the drug to modulate the activity of the neural adhesion molecule.
Yet another object of the invention is to provide an assay system for screening drugs and other agents for ability to modulate the production of a neural adhesion molecule, which includes the glial cell culture system; and CNS neurons added to the cell culture system.
A further object of the invention is to provide an assay system for screening drugs and other agents for ability to modulate the production of a neural adhesion molecule, which includes culturing the glial cell culture system inoculated with a drug or agent; adding CNS neurons to the cell culture system; and examining neuronal outgrowth to determine the effect of the drug thereon.
Yet another object of the invention is to provide an assay system for screening drugs and other agents for ability to modulate the production of a neural adhesion molecule, which includes culturing the optic nerve or other parts of the nervous system in the cell culture system inoculated with a drug or agent; adding CNS neurons to the cell culture system; and examining neuronal outgrowth to determine the effect of the drug thereon.
Another object of the invention is to provide an assay system for screening drugs and other agents for ability to modulate the production of a neural adhesion molecule, which includes inoculating a culture of CNS neurons with a drug or agent; adding a soluble neural adhesion molecule; and examining neuronal outgrowth to determine the effect of the drug thereon.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description taken with reference to the following illustrative drawings.